The present invention relates generally to DNA binding regulatory proteins and more particularly to DNA sequences encoding androgen receptor protein and novel DNA binding proteins designated TR2, to the polypeptide products of recombinant expression of these DNA sequences, to peptides whose sequences are based on amino acid sequences deduced from these DNA sequences, to antibodies specific for such proteins and peptides, and to procedures for detection and quantification of such proteins and nucleic acids related thereto.
There are five major classes of steroid hormones: progestins, glucocorticoids, mineralocorticoids, androgens, and estrogens. Receptor proteins, each specific for asteroid hormone, are distributed in a tissue specific fashion and in target cells, steroid hormones can form specific complexes with corresponding intracellular receptors. [Jensen, et al., Proc. Nat'l. Acad. Sci. (USA), 59:632 (1968); Gorski, et al., Ann. Rev. Physiol., 38:425-450 (1976); and Liao, et al., page 633 in Biochemistry of Hormones, H. L. J. Makin, ed. (Blackwell Sci. Publ. Oxford, 1984)]. The hormonal regulation of gene expression appears to involve interaction of steroid receptor complexes with certain segments of genomes and modulation of specific gene transcription. See, e.g., Ringold, Ann. Rev. Pharmacol. Toxicol., 25:529 (1985); and Yamamoto, Ann. Rev. Genet., 19:209 (1985). Many of the primary effects of hormones involve increased transcription of a subset of genes in specific cell types.
The successful cloning of e.g., cDNAs coding for various steroid receptors has allowed the structural and functional analysis of different steroid receptor domains involved in steroid and DNA binding. See, e.g., Hollenberg, et al., Nature (London), 318:635 (1985); Miesfeld, et al., Cell, 46:389 (1986); Danielsen, et al., EMBO J., 5:2513 (1986); Greene, et al., Science, 231:1150 (1986); Green, et al., Nature (London), 320:134 (1986); Krust, et al., EMBO J., 5:891 (1986); Loosfelt, et al., Proc. Nat'l. Acad. Sci. (USA), 83:9045 (1986); Conneely, et al., Science, 233:767 (1987); Law, et al., Proc. Nat'l. Acad. Sci. (USA), 84:2877 (1987); Misrahi, et al., Biochem. Biophys. Res. Commun., 143:740 (1987); Arriza, et al., Science, 237:268 (1987); Sap, et al., Nature (London), 324:635 (1986); Weinberger, et al., Nature (London), 318:641 (1986); Benbrook, et al., Science, 238:788 (1987); and Evans, Science, 240:889 (1988).
Androgens, such as testosterone, are responsible for the development of male secondary sex characteristics and are synthesized primarily in testis. Cloning of a cDNA for androgen receptor (AR) has been difficult because, until recently, monospecific antibodies against AR have not been available for screening cDNA libraries. An abstract by Govindan, et al., J. Endocrinol. Invest., 10 (Suppl. 2) (1987), reported the isolation of cDNA clones encoding human androgen receptor isolated from a human testis .lambda.gt-11 cDNA library using synthetic oligonucleotides homologous to human glucocorticoid, estradiol, and progesterone receptors as probes. The expressed protein reportedly bound tritium-labelled DHT (dihydrotestosterone) with high affinity and specificity. However, no nucleotide or amino acid sequence analysis was provided for full length androgen receptors, nor was any description provided concerning isolation of the full length putative androgen receptor clones.
Recently, Chang, C., et al., Science, 240:324 (Apr. 15, 1988), co-authored by the inventors herein, described cDNAs encoding androgen receptors obtained from human testis and rat ventral prostrate cDNA libraries. These cDNAs for human and rat androgen receptor were reported to be long enough to code for 94 kDa and 76 kDa receptors. The molecular weights were derived with the assistance of a software program known as: DNA Inspector II (Textco West Lebanon, New Hampshire) open reading frame analysis. With a new DNA Inspector IIe program, hAR (918 amino acids) has an estimated molecular weight 98,608 and rAR (902 amino acids) has a molecular weight of 98,133. Therefore, the reported "94 kDa" AR is now termed "98 kDa" AR; and the hAR or rAR polypeptides, from the second ATG/Met, reported as "76 kDa" are now termed "79 kDa". See also, Chang, C., et al., Proc. Nat'l. Acad. Sci. (USA), 85:7211 (Oct. 5, 1988) also co-authored by the inventors herein.
In contrast, Lubahn, D., et al., Science, 240:327 (1988), using libraries from human epididymis and cultured human foreskin fibroblasts obtained a human cDNA which was expressed in monkey kidney (COS) cells to yield a protein, present in the cytosol, capable of binding androgens. This cDNA, however, was only sufficient to-code for a receptor having an estimated molecular weight of 41,000. Therefore, the cDNA obtained only coded for a portion of AR.
Of interest to the present invention is Young, et al., Endocrinol., 123:601 (1988), wherein the production of anti-AR monoclonal antibodies was reported. Anti-AR autoantibodies were identified in the sera of prostate cancer patients, as described in Liao, S., et al., Proc. Nat'l. Acad. Sci. (USA), 82:8345 (1984) (one of the co-inventors herein), and were characterized with respect to their titer, affinity, and specificity. Subsequently, lymphocytes from the blood of those patients having high antibody titers were isolated, transformed with Epstein-Barr Virus (EBV), and cloned for anti-AR monoclonal antibody production. These monoclonal antibodies were found to interact with androgen receptors from rat prostate. An attempt to scale-up antibody production resulted in a decline of antibody secretion. It is not uncommon for transformed B-cells to be more unstable than hybridoma cells. Kozbor, et al., Eur. J. Immunol., 14, 23 (1984). Because of the instability associated with such cell lines, an alternate source of monoclonal antibodies is preferred.
There thus exists a need in the art for information concerning the primary structural conformation of androgen receptor protein and other DNA binding proteins such as might be provided by knowledge of human and other mammalian DNA sequences encoding the same. Availability of such DNA sequences would make possible the application of recombinant methods to the large scale production of the proteins in procaryotic and eukaryotic host cells, as well as DNA-DNA, DNA-RNA, and RNA-RNA hybridization procedures for the detection, quantification and/or isolation of nucleic acids associated with the proteins. Possession of androgen receptor and related DNA-binding proteins and/or knowledge of the amino acid sequences of the same would make possible, in turn, the development of monoclonal and polyclonal antibodies thereto (including antibodies to protein fragments or synthetic peptides modeled thereon) for the use in immunological methods for the detection and quantification of the proteins in fluid and tissue samples as well as for tissue specific delivery of substances such as labels and therapeutic agents to cells expressing the proteins.